We propose to renew funding for the National Research Resource for Imaging Mass Spectrometry (IMS) at Vanderbilt University School of Medicine, Nashville TN. The previous funding cycle has been extremely successful in both advancing IMS technology and applying it to cutting edge biological projects. In this renewal, we seek to advance the technology to provide next generation capabilities that will encompass sub-micron spatial resolution, ultra-high sensitivity, and ease of use tools that will be made availabl to the non-expert investigator. This proposal contains two overarching aims. First, it proposes to pioneer novel instrument technologies, sample preparation devices and methods, and data analysis/image informatics solutions. The latter is a critical need for current research grade IMS instrumentation, and it will be essential for next-generation instruments.. Second, this proposal seeks to simplify the entire imaging process that currently requires expertise and significant time to get high quality, meaningful results. Achievement of these global aims is important to bring this technology into the biological and clinical laboratory. The proposed Technology Research & Development (TRD) program will provide performance improvements that will meet many of the current needs of the user community. Specific TRD advancements will focus on maximizing instrument sensitivity and spatial resolution. We will also develop specialty reagents and methodologies designed to enhance sensitivity, molecular specificity, and preserve the location of biomolecules during sample preparation and analysis. We propose to develop a next generation imaging mass spectrometer for routine use that is capable of achieving 500 nm spatial resolution at high sensitivity. Additionally, we will develop software capable of co-registering and viewing imaging data from multiple modalities such as IMS and microscopy through an image fusion process. Finally, we will develop new advanced bio-computational algorithms to mine imaging data for molecular patterns and to normalize images from large datasets. These developing advanced technologies will be focused on important driving biomedical projects (DBPs) in infectious disease, eye disease, diabetes, and cancer. Important activities of the Resource include: selected collaborations with investigators who have NIH sponsored research projects that would significantly benefit from the developing IMS technology. This includes high spatial resolution imaging, high mass resolving power instrumentation and advanced data analysis and informatics capabilities. Training and dissemination of the technology will continue to be accomplished through a yearly on-site four day training course termed AIMS (Advanced Imaging Mass Spectrometry). In addition, training and dissemination will occur through hosting individual visiting investigators, students, and fellows, and through publication of the research outcomes for both the technology and its applications, and from individual invited research lectures presented by the faculty of the Resource.